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. 2014 Sep;9(9):1271-9.
doi: 10.4161/epi.32087. Epub 2014 Aug 1.

DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos

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DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos

Azizollah Bakhtari et al. Epigenetics. 2014 Sep.

Abstract

Dppa3 has been described in mice as an important maternal factor contributed by the oocyte that participates in protecting the maternal genome from oxidation of methylated cytosines (5mC) to hydroxymethylated cytosines (5hmC). Dppa3 is also required for normal mouse preimplantation development. This gene is poorly conserved across mammalian species, with less than 32% of protein sequence shared between mouse, cow and human. RNA-seq analysis of bovine oocytes and preimplantation embryos revealed that DPPA3 transcripts are some of the most highly abundant mRNAs in the oocyte, and their levels gradually decrease toward the time of embryonic genome activation (EGA). Knockdown of DPPA3 by injection of siRNA in germinal vesicle (GV) stage oocytes was used to assess its role in epigenetic remodeling and embryo development. DPPA3 knockdown resulted in increased intensity of 5hmC staining in the maternal pronucleus (PN), demonstrating a role for this factor in the asymmetric remodeling of the maternal and paternal PN in bovine zygotes. Also, DPPA3 knockdown decreased the developmental competence of parthenogenetic and in vitro fertilized embryos. Finally, DPPA3 knockdown embryos that reached the blastocyst stage had significantly fewer ICM cells as compared with control embryos. We conclude that DPPA3 is a maternal factor important for correct epigenetic remodeling and normal embryonic development in cattle, indicating that the role of DPPA3 during early development is conserved between species.

Keywords: 5-hydroxymethylcytosine; DPPA3; TET; epigenetic; knockdown; preimplantation; reprogramming; zygote.

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Figures

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Figure 1. Transcript abundance for DPPA3 and TET genes in bovine oocytes and preimplantation embryos. RPKM: reads per kilobase of gene model per million mapped reads. MII: metaphase II oocytes; 2-C: 2-cell embryos; 8-C: 8-cell embryos; 8-C+Aman: 8-cell embryos developed under the presence of the transcriptional inhibitor α-amanitin, Bl: blastocysts.
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Figure 2. Efficient knockdown of DPPA3 mRNA levels in MII oocytes by siRNA injection into GV oocytes. Levels of DPPA3 and TET2 mRNA in control and DPPA3 siRNA-injected oocytes 24 h after injection. a,b Columns with different letters indicate significant differences (P < 0.0001).
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Figure 3.DPPA3 knockdown in bovine oocytes alters the pattern of 5hmC staining in late PN stage embryos. (A) Representative immunofluorescence images of late PN stage embryos (22 h after in vitro insemination in bovine and PN5 stage in mouse). Scale bar = 50 µm. (B) Difference in fluorescence intensity between maternal and paternal PN within individual PN stage embryos (average intensity of paternal PN was subtracted from average intensity of maternal PN). a,b: Different letters indicate significant differences (P < 0.05).
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Figure 4.DPPA3 knockdown in bovine oocytes results in increased 5hmC in the female pronucleus. Average fluorescence intensity of 5mC and 5hmC immunostaining at paternal and maternal PN in zygotes derived from control and DPPA3 siRNA-injected oocytes. a,b: Different letters indicate significant differences (P < 0.05).
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Figure 5.DPPA3 knockdown reduced oocyte developmental competence after parthenogenetic activation or in vitro fertilization. In vitro developmental rates of parthenogenetic (A) and IVF-derived embryos (B) produced from control and DPPA3 siRNA-injected oocytes.
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Figure 6. Effect of DPPA3 knockdown in bovine oocytes on cell number and allocation of resulting blastocysts. Representative images of embryos immunostained for SOX2 and CDX2 as markers of ICM and TE cells, respectively. Nuclei were counterstained with Hoechst33342. Scale bar = 50 µm. (B) Average number of cells and ratio of ICM:TE cell number in embryos produced from control and DPPA3 siRNA-injected oocytes. ICM: inner cell mass; TE: trophectoderm. a,b: Different letters indicate significant differences (P < 0.05).

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